This revision application is submitted in response to NOT-OD-09-058: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications. The goal of the current grant is to understand the role of ribbon synapses in communicating visual information in the retina. Visual signals originate in the photoreceptor cells, which then communicate with second-order bipolar neurons via ribbon synapses. In turn, the bipolar neurons pass the signals on to third-order neurons in the inner retina, again via ribbon synapses. Ribbons are proteinaceous organelles at the active zones of photoreceptor and bipolar cell synapses, which are able to support high rates of neurotransmitter release for prolonged periods. It is not yet known how ribbons allow these synapses to sustain release in this way, while other synapses in the brain fatigue rapidly during steady stimulation. The existing aims of the project use a combination of cellular electrophysiology and high-resolution fluorescence imaging to obtain detailed information about the synaptic vesicle cycle at the specialized ribbon synapses of photoreceptors and bipolar cells. In the new specific aim introduced in the revised project, electron microscopy will be used to obtain complementary information about the ultrastructure of ribbon synapses that are rapidly frozen during ongoing neurotransmitter release. This expanded aim will provide an answer to the question of whether synaptic ribbons support sustained neurotransmitter release by allowing synaptic vesicles to fuse with other vesicles, that is, by compound exocytosis. The results will provide essential information about how visual signals are transmitted through the retina. PUBLIC HEALTH RELEVANCE: In a variety of degenerative diseases, blindness results from loss of the retina's photoreceptor cells, which convert light into electrical signals that are then relayed through synaptic connections to other neurons of the retina and ultimately to the brain, where they form the basis for visual perceptions. Current therapeutic strategies focus on restoring the photoreceptors'ability to convert light into electrical signals, but the transmission of those signals through the retina is equally important to vision. The goal of this project is to provide new and comprehensive understanding of this essential transmission process, including the underlying molecular machinery.